KR20180094125A - Mini-gastrin analogue, in particular for use in cck2 receptor positive tumour diagnosis and/or treatment - Google Patents

Abstract

It is an object of the present invention to provide a gastrin analog exhibiting high uptake by very low intracellular accumulation in CCK-2 receptor positive tumors at the same time. This object is according to the invention, the formula: has an X-DGlu-DGlu-DGlu- DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Y-Asp-Phe-NH 2, where Y is to replace the methionine Amino acid and X is achieved by the small-gastrin analog of PP-F11, which represents a chemical group attached to the peptide for the purpose of diagnostic and / or therapeutic intervention for CCK-2 receptor related diseases. In particular, a highly suitable compound in terms of high tumor to kidney ratio is a small-gastrin analog with six D-glutamic acid or six glutamine. These compounds are readily oxidizable and still retain methionine, a disadvantage to clinical applications under GMP due to the possible forms. Thus, the removal of methionine generally leads to a low oxidation affinity that favor tumor-kidney ratio. In a preferred embodiment of the present invention, methionine is substituted with norleucine. The so-called PP-F11N small-sized gastrin now represents the best tumor-kidney ratio and is therefore the most promising candidate for clinical applications.

Description

In particular, the use of small-gastrin analogs for the diagnosis and / or treatment of CCK2 receptor positive tumors,

The present invention relates to small-gastrin analogs and their use in the diagnosis and / or treatment of CCK2 receptor positive tumors.

G protein-coupled receptors (GPCRs) have been used as target proteins for radiolabeled peptides since the early 90s. The somatostatin receptor was a prototype of radio-nuclide imaging and treatment with peptides, leading to clinical first-line treatment of neuroendocrine tumors with Y-90 and Lu-177 labeled derivatives of octreotide ). Several radiolabeled peptides, including a gastrin-recognized peptide analog (GRP), a glucagon-like peptide 1 analog (GLP-1), a neurotensin analog (NT) or a neuropeptide Y analog (NPY) (Macke, Reubi J Nucl Med 2008; 49: 1735-1738). An additional very interesting target was the cholecystokinin-2 receptor (CCK-2R). This receptor is expressed primarily in thyroid carcinoma (MTC), small cell lung cancer (SCLC) and ovarian metastatic tumors (Reubi, Int J Cancer. 1996, and Reubi, Cancer Res. Radiolabeled gastrin analogs are good candidates for targeting imaging and therapy. In-111 labeled galactin analogs showed superior MTC detection compared to Octrecasc-111, providing additional information on neuroendocrine tumors, especially when they are negative in somatostatin receptor synthography (Endocr Relat Cancer. 2006 Dec; 13 (4): 1203-11.) And Eur J Nucl Med Mol Imaing 2006 Nov; 33 (11): 1273-9).

However, due to high renal absorption, radiolabeled peptides could not be used for therapy. High kidney absorption is associated with six negatively charged glutamate. Twelve kinds of gastrin-related compounds were designed, synthesized and compared as 111In labeled compounds. The best compound in terms of high tumor-to-kidney ratio is small-gastrin with six D-glutamic acid or six glutamine. These compounds still retain methionine that can be easily oxidized. This is a disadvantage in clinical applications because the receptor affinity is rapidly reduced after methionine oxidation and production under GMP can be abruptly inhibited.

The high likelihood of significant improvement in the treatment and imaging of patients with metastatic thyroid carcinoma (MTC), small cell lung cancer (SCLC) and further CCK-2 receptor positive tumors is due to the fact that tumor cells are specific for radiation- It is to cover. The basis of this finding is the evidence of overexpression of individual CCK-2 target receptors in 92% of the irradiated MTC, and this evidence has been obtained by in-vitro studies (Reubi 1997). Furthermore, the same study population identified identical overexpression of CCK-2 target receptors in 57% of small cell lung cancer, 65% of astrocytomas, and 100% of ovarian stromal tumors.

The first treatment study (phase 0 study) was conducted on 8 patients with advanced thyroid carcinoma. Partial grading was achieved in both patients, and four patients showed a stabilization of the previous strong progressive pathway of cancer disease MTC after treatment with ⁹⁰ Y-labeled small-gastrin analogs. This study had to be discontinued due to the renal toxicity of the treatment in terms of the accumulation in the intestinal kidney of the material used for the assay.

With the support of the European COST Program (European Science and Technology Cooperation), a number of significantly improved radiolabeled gastrin-analogs have been synthesized by various research groups and their characteristics have been investigated. Compared to conventional gastrin analogs, these new materials possess a significantly higher tumor to kidney ratio in terms of absorption in human tissues (Laverman 2011, Polenc-Peitl 2011, January 2011, [Fani 2012]). Among these novel gastrin analogues, ¹⁷⁷ Lu-PP-F11 (a linear small-gastrin analog having 6 D-Glu residues, hereinafter referred to as PP-F11), together with a high accumulation in its favored tumor, , Which demonstrated the best properties for future radionuclide therapy.

It is therefore an object of the present invention to provide a galactin analog that exhibits a much better accumulation in the CCK-2 receptor positive tumors as well as a very low accumulation in the kidney.

The purpose of the formula: have the X-DGlu-DGlu-DGlu- DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Y-Asp-Phe-NH 2, where Y represents an amino acid substitution of methionine and X is Galactin analog of PP-F11, which represents a chemical group attached to the peptide for diagnostic and / or therapeutic intervention with respect to CCK-2 receptor related diseases.

In particular, a very suitable compound in terms of high tumor to kidney ratio is a small-gastrin analog with six D-glutamic acids or six glutamines. These compounds are readily oxidizable and still retain methionine, a disadvantage in clinical applications under GMP due to the possible morphology. Thus, substitution of methionine by amino acids that are non-oxidative, such as stereoisomeric but not biologically active, results in compounds that do not have oxidative potential. This avoids oxidation during storage and production which can lead to low affinity compounds resulting in low tumor to kidney ratios.

In a preferred embodiment of the present invention, methionine is substituted by norleucine. This so-called PP-F11N small-sized gastrin now represents the best tumor-kidney ratio and is therefore the most promising candidate for clinical applications. In terms of radiation cancer therapy, X is a radionuclide comprising a radiator such as a chelator for a non-metallic sub-atomic group such as a radioactive metal, e.g., ¹⁷⁷ Lu, or ⁹⁰ Y, or ¹¹¹ In, or F-18 or radioactive iodine . To improve medical imaging, X is optically active chemicals, for example, Alexa fluorine (Alexa Fluor) ^® 647, ahyial die (IRDye) 680RD or DY-700 be for, and photo print (Photofrin for the optical therapeutic application ), Forscam or Photochlor. ≪ / RTI > For both applications, the active chemical may be an optically active nanoparticle. To support chemotherapeutic intervention, X may represent a chemotherapeutically active compound, such as gemcitabine, doxorubicin or cyclophosphamide. Delivery of the formulations described can be done by nanoparticles or liposomes as X, which are loaded with the chemotherapeutic agent.

In view of the use of the small gastrin of the present invention, diagnostic intervention for CCK-2 receptor related diseases and / or therapeutic intervention for CCK-2 receptor related diseases is anticipated.

BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will be described in more detail in the context of the accompanying drawings, in which: Fig.
Fig. 1 Structural design of PP-F11N starting with the small-gaestrin analog PP-F11 from the COST scheme;
Figure 2 Four hours post-biodistribution in CD1 nu / nu mice model of PP-F11, PP-F11N and PP-F11 ox (oxidized PP-F11);
Figure 3 Four-hour post-biodistribution in the CD1 nu / nu mouse model of PP-F10, PP-F10N and PP-F10 ox (oxidized PP-F10); And
Figure 4 Stability of various small-gastrin analogs over time

Figure 1 shows the mini-galactin analogue PP-F11 derived from the COST scheme mentioned above. The modified small-gastrin analog PP-F11N is achieved by exchanging the oxidizable amino acid methionine with norleucine. DOTA represents 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid which is an organic compound of the formula (CH ₂ CH ₂ NCH ₂ CO ₂ H) ₄ . This molecule consists of a central 12-member tetraaza (i.e., contains four nitrogen atoms) rings. DOTA is a complexing agent, especially for lanthanide ions. Its complexes have contrasting agents, and medical applications as cancer treatments.

PP-F11N was investigated according to the CD1 nu / nu mouse model. Compared with PP-F11, the mini-gastrin analog PP-F11N exhibits significantly higher tumor uptake, which also leads to a very favorable tumor-to-noise ratio with very little intimal accumulation. Figure 2 shows the biodistribution of PP-F11, PP-F11N and oxidized PP-F11 ox (oxidized PP-F11) in the athymic CD1 nu / nu mouse model after 4 hours. Tumor positive: A431 cells transfected with human CCK-2 receptor on one side of the mouse; Tumor negative: CCK-2 receptor negative A431 cells on the other side of the mice. The effect of high tumor uptake by replacement of methionine with norleucine is particularly pronounced in compounds with the DGlu6 sequence, unlike compounds with the DGLn6 sequence, designated PP-F10.

FIG. 3 shows the results for PP-F10, PP-F10N and PP-F10 ox (oxidized PP-F10) compared to the results shown in FIG. The structural formula of PP-F10 is given below:

PP-F10: DOTA-DGln- DGln-DGln-DGln-DGln-DGln-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2

PP-F10N: DOTA-DGln- DGln-DGln-DGln-DGln-DGln-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2

PP-F10 ox: DOTA-DGln -DGln-DGln-DGln-DGln-DGln-Ala-Tyr-Gly-Trp-Met (ox) -Asp-Phe-NH 2

Promising tumor uptake is therefore not visible in Figure 3, as other organs such as the eye, kidney, and bone have a much higher dose than the target tumor.

Figure 4 shows the pharmacological stability of PP-F11N (all ¹⁷⁷ Lu radiolabeled) compared to PP-F11, PP-F10N and PP-F10. Stability was confirmed in human serum; Measurement of the metabolites was carried out by means of HPLC. The small-gastrin analog PP-F11N according to the present invention has the highest pharmacological stability at all feet.

Materials: Peptides (PP-F10, PP-F10N, PP-F11 and PP-11N) were synthesized by PLS (Heidelberg, Germany) by the Merrifield method. All chemicals were purchased from Sigma-Aldrich (Buick, Switzerland). A431 cells (squamous cell carcinoma cell lines) were stably transfected with CCK2R-encoding cDNA or empty vector (false-transfection) and were donated from ¹ L-Aloj (Napoli). Lu-177 was purchased from ITG (Munich, Germany). The peptide conjugate was complexed with natural ^nat Lu.

Labeling of small-gastrin was performed in the following environment.

System for HPLC analysis:

System: Pump Varian Prostar 2030.01, Diode Array 330.71, Autosampler 410, Packard Radiomatic Flow-One \

Column: Stability 120 BS-C23 3 μm 150 * 4.6 mm, Dr. Maisch Gradient:

System for purification:

Pump 1: Waters 515, Pump 2: Hitachi L-7000, Knauer UV detector K2510, Radio-Monitor Eberline, interface SS420X, EZstart

Rheodyne manual injector

Column 1: Stability 120 BS-C23 3 μm 10 * 4.6 mm,

Column 2: Stability 120 BS-C23 3 [mu] m 150 * 4.6 mm,

product:

Lu-177: Product number Lu-12-052-01 / 121042, activity 2 GBq / 200 μl 0.04 M HCL, ITG (ITM AG)

PP-F11N: 0.25 mM H20 solution

Ammonium solution: Sigma-Aldrich metal free

Na ascorbate: Sigma-Aldrich

HCl 30%: Sigma-Aldrich metal-free

H20: obtained from Milipore system Biotel

Peptide PPF11N is labeled with Lu-177:

PPF-11N was labeled with Lu-177 at an isotope: peptide ratio of 1:47.

Lw binding of a mixture of Lu-177 and peptide to 1.5 mL of Eppendorf:

                                    - 20 μl Lu-177 (190 MBq)

                                    - 5 μl ammonium ascorbate 0.7 M

                                    - 50 [mu] l PPF-11N 0.25 mM

                                    - 5 μl HCl 0.04 M

The mixture was heated at 95 < 0 > C for 20 minutes.

The complex was then purified and confirmed by HPLC.

Both syntheses were achieved simultaneously.

Purification of the labeled peptides by HPLC

Both label reactions were injected into 2 D HPLC.

Description of 2D HPLC:

Step 1: The product is injected into the loop with a leodane manual injector and the product is pushed through column 1 to the first pump. The product was transferred from the rope to column 1 and washed with H2O + 0.1 TFA.

Step 2: Start the gradient with the second pump and change the valve position to connect column 1 and column 2 in series.

Push the product from Column 1 to Column 2. Excess peptides are separated from labeled Lu-177-PPF11N in column 2 and collected in fractions of 500 ul size. The collected tubes still contain 5 mg of Na-ascorbate.

Purification results by HPLC

Preparation of labeled peptides for intravenous (i.v.) injection of mice

The two fractions were fused and the solvent was evaporated for 35 minutes.

Then, 600 ul PBS 1x was added with 10 ul 5 mM DTPA solution.

Final solution: 295 MBq / 610 ul

stability

12 MBq of radiolabeled compound was cultured in 2 mL of fresh human plasma. 40 μL detectors were harvested at 0, 1, 2, 18, 24, 48, and 72 hours later and 200 μL (50% methanol and 50% acetonitrile) was added to the Mini-UniPrep filter. The solution was vortexed and then filtered. 40 [mu] L of the filtered solution was analyzed by HPLC.

Biodistribution study

CD1 nu / nu mice were injected with 5x10 ⁶ A431 cells. CCK-2 receptor positive A431 cells ¹ were injected on one side, and false cells as an unspecified control on the other. The tumors reached a weight of about 80 to 120 mg after about 10 days. 150-200 kBq (5 pmol) of radiolabeled peptide was injected into the tail vein. The mice died from CO ₂ suffocation after a defined time point after injection. Organs were excised, weighed and their activity was measured. The% injected activity per gram (% iA / g) was calculated. Animal testing was approved by the local animal welfare committee and carried out in accordance with national regulations.

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Claims (15)

Small-gastrin analogs of the formula:

here,
Y represents an amino acid substituting methionine,
X represents a chemical group attached to the peptide for diagnostic or therapeutic intervention with CCK-2 receptor related diseases. 2. The small-gastrin analog according to claim 1, wherein Y is norleucine. 2. The small-gaustin analog according to claim 1, wherein X represents a radionuclide comprising a chelator for the radiation metal. The small-gastrin analog according to claim 3, wherein the chelator for the radiation metal is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The small-gastrin analog according to claim 3, wherein the radionuclide is selected from the group consisting of ¹⁷⁷ Lu, ⁹⁰ Y and ¹¹¹ In. 5. The small-gaustrin analog according to claim 4, wherein the radionuclide species is selected from the group consisting of ¹⁷⁷ Lu, ⁹⁰ Y and ¹¹¹ In. 3. The method of claim 2,

Lt; RTI ID = 0.0 &gt; gastrin &lt; / RTI &gt; 8. The method of claim 7,

This ¹⁷⁷ Lu-labeled mini-gastrin analogue. 2. The small-gaustrin analog according to claim 1, wherein X represents a radionuclide, an optically active chemical compound, a chemotherapeutically active compound, a nanoparticle or a liposome. 10. The small-gaustrin analog according to claim 9, wherein X represents a radionuclide comprising a sub-atomic group for a non-metal. 10. The small-gastrin analog according to claim 9, wherein X represents an optically active chemical compound that is a photosensitizer or an MRI contrast agent. 10. The small-gastrin analog according to claim 9, wherein X represents a chemotherapeutically active compound that is gemcitabine, doxorubicin or cyclophosphamide. 10. The small-gaustrin analog according to claim 9, wherein X represents a nanoparticle or liposome with a radionuclide or an optically active or MRI contrast agent. 13. A diagnostic agent for a CCK-2 receptor related disease, comprising a small-gastrin analogue according to any one of claims 1 to 13. 14. A therapeutic agent for a CCK-2 receptor related disease, comprising a small-gastrin analogue according to any one of claims 1 to 13.

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